Zhifei Li

Gene Expression Profiling of Grass Carp (Ctenopharyngodon idellus) and Crisp Grass Carp.

Grass carp (Ctenopharyngodon idellus) is one of the most important freshwater fish that is native to China, and crisp grass carp is a kind of high value-added fishes which have higher muscle firmness. To investigate biological functions and possible signal transduction pathways that address muscle firmness increase of crisp grass carp, microarray analysis of 14,900 transcripts was performed. Compared with grass carp, 127 genes were upregulated and 114 genes were downregulated in crisp grass carp. Gene ontology (GO) analysis revealed 30 GOs of differentially expressed genes in crisp grass carp. And strong correlation with muscle firmness increase of crisp grass carp was found for these genes from differentiation of muscle fibers and deposition of ECM, and also glycolysis/gluconeogenesis pathway and calcium metabolism may contribute to muscle firmness increase. In addition, a number of genes with unknown functions may be related to muscle firmness, and these genes are still further explored. Overall, these results had been demonstrated to play important roles in clarifying the molecular mechanism of muscle firmness increase in crisp grass carp.

Proteomic signature of muscle fibre hyperplasia in response to faba bean intake in grass carp

Fish muscle growth is important for the rapidly developing global aquaculture industry, particularly with respect to production and quality. Changes in muscle fibre size are accomplished by altering the balance between protein synthesis and proteolysis. However, our understanding regarding the effects of different protein sources on fish muscle proteins is still limited. Here we report on the proteomic profile of muscle fibre hyperplasia in grass carp fed only with whole faba bean. From the results, a total of 99 significantly changed proteins after muscle hyperplasia increase were identified (p < 0.05, ratio <0.5 or >2). Protein–protein interaction analysis demonstrated the presence of a network containing 56 differentially expressed proteins, and muscle fibre hyperplasia was closely related to a protein–protein network of 12 muscle component proteins. Muscle fibre hyperplasia was also accompanied by decreased abundance in the fatty acid degradation and calcium signalling pathways. In addition, metabolism via the pentose phosphate pathway decreased in grass carp after ingestion of faba bean, leading to haemolysis. These findings could provide a reference for the prevention and treatment of human glucose-6-phosphate dehydrogenase deficiency (“favism”).

Microbial succession in biofilms growing on artificial substratum in subtropical freshwater aquaculture ponds

Abstract Biofilms can be used to improve the water quality in aquaculture ponds, and elucidating the process of microbial succession in biofilms would allow the characterization of metabolic processes and permit optimization. In the present study, microbial succession of a biofilm growing on artificial substrata in a subtropical freshwater pond was investigated by high‐throughput sequencing. Providing artificial substrata effectively reduced the concentrations of total nitrogen and total phosphorus in the pond. Relatively stable microbiota were formed after approximately 1 week. The dominant phyla in the mature biofilm were Proteobacteria, Bacteroidetes, Firmicutes and Actinobacteria. The relative abundances of denitrifiers and phosphorus‐removing bacteria, such as those in Comamonadaceae and Neisseriaceae, were significantly increased. The use of avermectin B1 changed the community structure of the microbiota; the microbiota were more similar to those at Week 0 than to those at Week 3. However, the microbial community structure recovered after approximately 1 week. Our results indicate that using artificial substrata can create a habitat for denitrifiers and phosphorus‐removing bacteria, and thereby improve pond water quality. This study provided insight into how the use of artificial substrata could improve water quality and elucidated the environment‐biofilm relationship in a subtropical freshwater pond.

Denitrification potential evaluation of a newly indigenous aerobic denitrifier isolated from largemouth bass Micropterus salmoides culture pond

This work evaluates the application potential of a new indigenous aerobic denitrifier, strain Pseudomonas CW-2, isolated from a largemouth bass culture pond. The rate of ammonium-N removal by strain CW-2 was approximately 97% at a DO concentration of 5.2 mg/L. Furthermore, when nitrate and ammonia coexisted, the strain gave priority to assimilating ammonia, and thereafter to denitrification. Under optimal cultivation conditions, citrate and acetate were the carbon resources, C/N was 8, dissolved oxygen was 5.2 mg/L, and pH was 7; the removal rate of ammonium reached nearly 90%. The changing patterns of different bacteria in strain CW-2-treated and the control pond water were also compared. Lower levels of ammonia, nitrite, and phosphates were observed in the treated water as compared with the controls. Meanwhile, phylum-level distributions of the bacterial OTUs revealed that Proteobacteria, Bacteroidetes, Planctomycetes, and Nitrospirae continuously changed their relative abundances in relation to carbon and the addition of strain CW-2; this finding implies that the conventional denitrification process was weakened under the effects of carbon or the presence of strain CW-2. We propose that strain CW-2 is a promising organism for the removal of ammonium in intensive fish culture systems, according to our evaluations of its denitrification performance.

Identification and expression analysis of miRNA in hybrid snakehead by deep sequencing approach and their targets prediction

MicroRNAs (miRNAs) play important regulatory roles in numerous biological processes, but there is no report on miRNAs of hybrid snakehead. In this study, four independent small RNA libraries were constructed from the spleen, liver kidney and muscle of hybrid snakehead. These libraries were sequenced using deep sequencing technology, as result, a total of 1,067,172, 1,152,002, 1,653,941 and 970,866 clean reads from these four libraries were obtained. 252 known miRNAs and 63 putative novel miRNAs in these small RNA dataset were identified. The stem-loop RT-qPCR analysis indicated that eight known miRNAs and two novel miRNAs show different expression in eight different kinds of tissues. For better understanding the functions of miRNAs, 95,947 predicated target genes were analyzed by GO and their pathways, the results indicated that these targets of the identified miRNAs are involved in a broad range of physiological functions.

Identification and comparative analysis of the miRNA expression profiles from four tissues of Micropterus salmoides using deep sequencing

In the present study, four small RNA libraries were constructed from an M. salmoides population and sequenced using deep sequencing technology. A total of 9,888,822; 8,519,365; 20,566,198; and 15,762,254 raw reads representing 666,097; 755,711; 978,923; and 840,175 unique sequences were obtained from the spleen, liver, kidney, and muscle libraries, respectively. As a result, 509 known miRNAs belonging to 143 families and 1157 novel miRNAs were identified. The miRNAs displayed diverse expression levels among the four libraries, among which most of the known miRNAs were expressed at higher levels than the novel miRNAs. Furthermore, stem-loop qRT-PCR was applied to validate and profile the expression of the differentially expressed miRNAs in the four different tissues, which revealed that some miRNAs showed tissue specific expression. The identification of miRNAs in M. salmoides will provide new information and enhance our understanding of the functions of miRNAs in regulating biological processes.

Smad4-dependent regulation of type I collagen expression in the muscle of grass carp fed with faba bean

Smad4 is the key regulator in the transforming growth factor β1 (TGF-β1)/Smads signal pathway, and is also the crux of the regulation of type I collagen expression in mammals. In fish, however, the relationship between Smad4 and type I collagen is still unknown. Given the widely accepted importance of type I collagen in fish muscle hardness, we seek to explore this issue by analyzing the expressions of the TGF-β1/Smads pathway molecules and type I collagen in the muscle of crisp grass carp fed with faba bean, which shows increased muscle hardness. The study found that (1) in the process of feeding the grass carp with faba bean, the mRNA and protein expressions of TGF-β1, Smad2 and Smad4 all increased along with the increase of type I collagen expression (Col1α1 and Col1α2); (2) one day after the injection of Smad4 over-expression vector, both mRNA and protein expressions of Col1α1 and Col1α2 significantly increased, reaching the maximum on the 2nd and 5th day, respectively; (3) one day after the injection of Smad4 RNAi interference vector, the mRNA and protein expressions of Col1α1 and Col1α2 decreased, reaching the minimum on the 5th day. These results revealed that Smad4 is the major regulator of type I collagen in the muscle of grass carp fed with faba bean. This study would provide an important mechanistic basis for nutritional regulation of type I collagen in the muscle of fish.

Broad Bean (Vicia faba L.) Induces Intestinal Inflammation in Grass Carp (Ctenopharyngodon idellus C. et V) by Increasing Relative Abundances of Intestinal Gram-Negative and Flagellated Bacteria

Constant consumption of broad bean (Vicia faba L.) induces intestinal inflammation and reduces growth rate in grass carp (Ctenopharyngodon idellus C. et V). However, the mechanisms underlying these effects are unclear. In mammalian models of inflammatory bowel disease (IBD), endotoxin and flagellin cause intestinal inflammation through upregulation of tumor necrosis factor (TNF)-α expression. We therefore speculated that broad bean consumption alters intestinal microbiota composition, thereby increasing the relative abundance of endotoxin-producing Gram-negative and flagellated bacteria and resulting in upregulation of TNF-α and intestinal inflammation in grass carp. We tested this hypothesis by comparing intestinal microbiota compositions of grass carp fed broad bean (GCBB), hybrid giant napier (Pennisetum sinese Roxb, GCHG), or formula feed (GCFF) by 16S rRNA gene sequencing. We also performed a histological analysis of the intestinal inner wall by scanning electron microscopy and measured intestinal wall and serum concentrations of TNF-α. Our results revealed epithelial cell damage including microvillus effacement and synechia along with increased TNF-α levels in the intestinal wall in the GCBB group as compared to the GCHG and GCFF groups. The relative abundances of Gram-negative and flagellated bacteria were also higher in the GCBB group than in the GCHG and GCFF groups; this was accompanied by upregulation of genes expressing endotoxin and flagellin in intestinal microbiota. Thus, broad bean-induced intestinal inflammation in grass carp shares features with IBD. Our findings demonstrate that the microbiome in fish is directly influenced by diet and provide a reference for deconstructing host–intestinal microbiota interactions.

The complete mitochondrial genome of Carassius auratus var. high back crucian carp (Cypriniformes: Cyprinidae)

Abstract In this study, the complete mitochondrial genome sequence of Carassius auratus var. high back crucian carp (C. auratus var.) was determined (GenBank accession number is KT756205). The mitochondrial genome sequence of C. auratus var. was a circular molecule with 16 581 bp in length, and contained 37 typical animal mitochondrial genes, including 2 ribosomal RNA genes, 13 protein-coding genes, 22 transfer RNA genes and a control region (D-loop). Four nucleotide compositions and their relative proportions of the entire mitogenome was 26.19% C, 16.06% G, 31.57% A and 26.18% T, with contents of A + T and G + C being 57.75% and 42.25%, respectively.

The complete mitochondrial genome of Shizothorax grahami (Cypriniformes: Cyprinidae)

Abstract Shizothorax grahami (S. grahami) is an underlying economic cold-freshwater fish in southwest China. In this study, the complete mitochondrial genome of S. grahami was determined (GenBank accession number is NC_029708). The mitochondrial genome sequence of S. grahami was a circular molecule with 16,584 bp in length, and contained 37 typical animal mitochondrial genes including 2 ribosomal RNA genes, 13 protein-coding genes, 22 transfer RNA genes and a control region (D-loop). Four nucleotide compositions and their relative proportions of the entire mitogenome was 27.14% C, 19.93% G, 29.58% A and 25.34% T. Among most species of genus Shizothorax, Schizothorax davidi had the closest relationship with S. grahami, and then Schizothorax prenanti.